3.1 4´-Aminomethyl-2,2´-bipyridyl-4-carboxylic acid (Abc) ruthenium conjugate Tris-diimine metal complexes of 4´-aminomethyl-2,2´-bipyridyl-4-carboxylic acid (Abc) are of interest, since they possess a number of favourable properties including high stability, inertness to ligand exchange reactions, tuneable electronic structures, long lifetimes in fluid solution, and high quantum yields. Site-specific labeled ruthenium oligonucleotides were prepared by DNA solid-phase synthesis using a ruthenium-nucleoside phosphoramidite,35 but this example lies not in the scope of this review. Another approach used bipyridyl amino acids and in particular Boc and Fmoc-protected Abc, which were incorporated into a hexapeptide.36
N
Scheme 17. Synthesis and metal complexation of Abc 74 and Boc/Fmoc protected derivatives.
Solid-phase synthesis of these metallopeptides was performed on MBHA resin using BOP and ByBOP as coupling reagents to provide high-affinity binding sites for ruthenium(II). Metal complexation occurred in solution followed by cleavage of the peptide from the solid support. The Abc residue bears the bipyridyl group not in a side chain but in the main peptide chain and is used as a tetradentate ligand to octahedrally coordinate and asymmetrically encapsulate a ruthenium(II) ion, creating a novel
peptide-caged redox-active metal complex. To prepare the Abc 74, a dual oxidation strategy was employed (Scheme 17). First, 4,4´-dimethyl-2,2´-bipyridine 70 was selectively oxidized to the 4´-monocarboxylic acid derivative 71. Second, the 4´-methyl group of 71 was oxidized with excess selenium dioxide to the aldehyde acid 4´-formyl-2´2-bipyridine-4-carboxylic acid 72. Oxime formation with hydroxyl-amine in ethanol/pyridine smoothly converted 72 into compound 73. Lastly, oxime acid 73 was transformed into the desired amino acid Abc 74 by catalytic hydrogenation. Amino acid 74 was converted into both Boc and Fmoc-derivatives for use in solid-phase peptide synthesis. Treatment of the Abc•HCl salt with di-(tert-butyl)dicarbonate provided Boc-Abc-OH 75 and similarly the reaction of Abc•HCl with Fmoc-succinimide furnished Fmoc-Abc-OH 76. The metal complexation properties of bipyridyl solid-phase peptide synthesis of building blocks 75 and 76 were confirmed by the synthesis of their respective ruthenium(II) octahedral mixed-ligand complexes. Reaction of 75 and 76 with dichlorobis(2,2´-bipyridine)ruthenium(II) (Rub2Cl2) gave the bis-heteroleptic complexes 77 and 78. To demonstrate the utility of Abc 74 in solid-phase peptide synthesis, a heptapeptide containing two Abc residues was synthesized to serve as a tetradentate caging peptide ligand for ruthenium(II) ions (Scheme 18). Two aminohexanoic acid residues (Ahx) were arranged as a bridging tether just long enough to form cis-bridged meridonal metal complexes. The C-terminal Gly residue was included to facilitate attachment of the bipyridine 77 or 78 to the sterically hindered MBHA resin, since direct coupling of Abc-OH to MBHA resin proved sluggish.
N
Scheme 18. Preparation of heteroleptic tris(bipyridyl) complex RuII(Aha)(bpy).
The acetylated hexapeptide amide Aha 79 was prepared by Boc/TFA strategy from Boc-Abc-OH 75 and other Boc amino acids using conventional reagents and procedures for manual solid-phase peptide synthesis. Coupling times and yields of 77 to the Gly-MBHA resin were remarkably improved by addition of stoichiometric amounts of the acylation catalyst DMAP. Following the assembly, apopetide 79 was cleaved from the resin with anhydrous HF and subsequent conversion of RuII(Aha)Cl2 to the heteroleptic tris(bipyridyl) complex RuII(Aha)(bpy) 80 was performed in solution.
3.2 Metallocene (ferrocene)37 conjugate
Ferrocene-containing tripeptides with one or two ferrocene building blocks were prepared by solid-phase peptide synthesis.38 Heinze et al. incorporated the solid-phase peptide synthesis-compatible ferrocene building block Fmoc-protected 1´-aminoferrocene-1-carboxylic acid (Fca)39 into the backbone of tripeptides. The coupling was performed using DIC/HOBt for activation and TentaGel-Wang, which turned out to be superior to polystyrene/divinyl resin, as solid support. Cleavage of the resulting tripeptides from the support with trifluoroacetic acid gave the mono- (Scheme 19) or diferrocene peptides. Reversible on-bead oxidation allows switching between the neutral ferrocene (low-affinity state) and charged ferrocenium ion (high affinity state), which results in superior anion-binding affinities.
O N
X = CH2,CHCH3,1,1´-ferrocenediyl
Scheme 19. Synthesis of ferrocene-containing tripeptides with ferrocene building block via solid-phase peptide synthesis.
Metallocene-modified tri- to penta-peptides were identified to have antibacterial activities,40 although the highest activity is still one order of magnitude lower than the minimum inhibitory concentration (MIC) values found for most naturally occurring antimicrobial peptides (AMPs). First Metzler-Nolte and co-workers synthesized metallocene-peptide bioconjugates where the amino acid sequence ranged from three to five residues by solid-phase peptide synthesis. The ferrocene and the cobaltocenium groups were introduced at the N-terminus by reacting ferrocene carboxylic acid hexafluorophosphate with the free amino group of the peptide 87, while the peptide was attached to the solid support. Attention has to be taken during the cleavage from the Rink amide resin. Decomposition, that is loss of a ferrocenoyl moiety, occurs when TFA/H2O/TIS cleavage mixture is used. However, this problem can be circumvented by the use of phenol rather than water.
Fmoc Rink
1) Fmoc -Deprotection
H Rink
2) Coupling to amino acid
Phe Rink Fmoc
3) Repeat steps 1 and 2 three times
Phe Rink Trp
Fmoc Arg Arg
4) Fmoc-Deprotection 5) Coupling to metallocene
Phe Rink Trp Arg Arg
O Fe
Phe NH2 Trp Arg Arg
O Fe 6) Cleavage and
side-chain deprotection
86 87 88
89
90 91
Scheme 20. Solid-phase peptide synthesis of metallocene-peptide bioconjugates.
Later, Metzler-Nolte and co-workers hoped to arrive at small, readily available artificial AMPs with activity comparable to the best naturally occurring AMPs by adding metallocenes to more active peptide sequences.41 Arg- and Trp-containig hexapeptide sequences which were shown to have good antibacterial properties42 were selected and modified by replacing the N-terminal amino acid with a ferrocenyl (and a cobaltocenium) group. The metallocene peptide conjugates were prepared on Rink amide resin whereas the ferrocene carboxylic acid was attached by forming an amide bond with the free N-terminal amino group of the solid support. The ferrocene moiety is stable towards deprotection reagents and to resin cleavage, however, the ferrocenoyl
Indeed, the activity of the resulting metallocene-pentapeptide conjugate [Fe(Cp)(C5H4 )-C(O)-WRWRW-NH2] 93 increased and was even better than 20 amino acid naturally occurring pilosulin, which was used as a positive control.
M H
N N
H H
N N
H H N O
O
O O
O
NH2
O
NH NH2 HN
NH NH2 HN
NH NH2 HN
HN NH
HN NH
HN O
O
O O
O
NH2 O
HN HN
NH NH2 HN
NH NH2 HN
HN HN HN
M
92 93
M = Co+, Fe
Figure 4. Metallocene-pentapeptide conjugate 92 and 93.